The present invention relates to the construction of a new class of Targeted Secretion Inhibitors (TSIs), to a method for the activation thereof, and to the activated product.
Non-cytotoxic proteases are a well-recognised group of proteases, which act on target cells by incapacitating cellular function. Importantly, non-cytotoxic proteases do not kill the target cells upon which they act. Some of the best known examples of non-cytotoxic proteases include clostridial neurotoxins (e.g. botulinum neurotoxin, which is marketed under names such as Dysport™, Neurobloc™, and Botox™) and IgA proteases.
Non-cytotoxic proteases act by proteolytically-cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle formation, and thus to secretion of molecules via vesicle transport from a cell. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell.
Non-cytotoxic proteases may be employed in their native or substantially native forms (i.e. as holotoxins, such as is the case with Dysport™, Neurobloc™, and Botox™), in which case targeting of the proteases to specific cell-types is reliant on (i) localised administration of the protease and/or (ii) the inherent binding ability of the native protease. Alternatively, non-cytotoxic proteases may be employed in a re-targeted form in which the native protease is modified to include an exogenous ligand known as a Targeting Moiety (TM). The TM is selected to provide binding specificity for a desired target cell, and, as part of the re-targeting process, the native binding portion of the non-cytotoxic protease may be removed.
The present Applicant has pioneered the concept and development of clostridial neurotoxin-based re-targeting technology, and the resulting fusion proteins are known as Targeted Secretion Inhibitors (TSIs).
TM replacement may be effected by conventional chemical conjugation techniques, which are well known to a skilled person. In this regard, reference is made to Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press.
Chemical conjugation is, however, often imprecise. For example, following conjugation, a TM may become joined to the remainder of the conjugate at more than one attachment site. Chemical conjugation is also difficult to control. For example, a TM may become joined to the remainder of the modified toxin at an attachment site on the protease component and/or on the translocation component. This is problematic when attachment to only one of said components (preferably at a single site) is desired for therapeutic efficacy. Thus, chemical conjugation results in a mixed population of modified toxin molecules, which is undesirable.
As an alternative to chemical conjugation, TM replacement may be effected by recombinant preparation of a single-chain polypeptide fusion protein. The preparation of such molecules is described in WO98/07864. However, the present inventors have identified that the WO98/07864 methodology is not suitable for all types of TM.
An alternative system to that of WO98/07864 is described in WO2006/059093. According to WO2006/059093, the TM is centrally-presented (CP) within the single-chain fusion protein, between the non-cytotoxic protease component and the translocation domain component. This results in a single-chain fusion protein having the following structure:NH2-[protease component]-[TM]-[translocation component]-COOH
The above-described fusion proteins are activated by treatment with a protease, which cleaves at a site located at the C-terminus of the protease component. This activation process results in a di-chain protein comprising the protease component attached covalently (via a disulphide linkage) to the translocation component of the fusion protein. In the case of WO2006/059093, the resulting di-chain molecule has a TM that is peptide-bonded via its C-terminus to the N-terminus of the translocation domain component. Accordingly, the N-terminal portion of the TM is then free to interact and bind to a desired receptor. This arrangement is important for the class of TMs that requires a free N-terminus or a free N-terminal portion in order to bind to its receptor.
By way of example, following proteolytic activation, WO2006/059093 provides polypeptides having the following di-chain conformation:

In said di-chain conformation, the TM and translocation components are presented in the form of a single-chain fusion protein, wherein the C-terminus of the TM is peptide-bonded to the N-terminus of the translocation component.
The present inventors have found that the systems described in WO98/07864 and WO2006/059093 are not optimal for the presentation of all types of TM, and, as such, may result in the production of fusion proteins having undesirable/reduced binding ability for the intended target cell.
There is therefore a need for an alternative or improved system for constructing TSIs.